PROJECTSUMMARY Tauisamicrotubule-stabilizingproteinthatisabundantinneurons.Itisahighlysoluble,intrinsicallydisordered protein(IDP)withlittletendencyforaggregationundernativeconditions.However,underseveralexperimental conditionsandinavarietyofneurodegenerativedisordersincludingAlzheimer?sdisease,Taucanspreadfrom cell to cell and aggregates as intra-cellular ?-sheet fibrilar deposits. Our laboratories have critical new data concerningthetemporal,structuralandcellbiologicaldetailsofTaumisfoldingandfluid-phaseassembly?the basis of this proposal. Our research team consists of a cell biologist, a physical chemist, and a theoretical biophysicist. Working together closely in an iterative mannerwe intend to determine the pathway from normal Tautodisease-relatedTaufibrils.Thetoolsforthisanalysisinclude(a)cellularsystemscapableofaddressing invivoTauinteractions,andindirectlyitsconformationalstatebasedonavarietyofmolecularprobes;?(b)site- directedspinlabeling,electronparamagneticresonance(EPR)lineshapeanalysisandpulseddipolarEPRto determine conformational signatures of Tau;? and (c) fully atomistic modeling of IDP conformations, their populationsandenergetics,andcoarse-grainedsimulationofhigher-orderassembliesofTau.Theconceptual flowoftheproposalbeginswitharemarkableobservationfromtheHanlab:Whenexposedtosub-stoichiometric amounts of heparin, segments of Tau dramatically extend by a nanometer to solvent-expose the hydrophobic PHF6(*) segment capable of stacking into neat ?-sheets. This observation correlates with the appearance of fibrils,andthuswerefertothisinitiatingstepas?onpathway?seeding.Invivo,Tauisknowntopopulateavast conformationallandscapecontrolledbyalternativesplicing,mutationsandpost-translationalmodifications.We propose that the IDP Tau populates an ensemble of different conformations with different aggregation propensities, fibril morphologies and interaction partners, depending on the exact Tau variant. However, the defining and specific conformational signatures within this ensemble are unknown. Determining the conformationalsignaturesofaggregation-proneTauvariantsisourcoreobjective,whileamissingpuzzlepiece inconnectingTauconformationtocellularinteractionsistheexistenceandnatureofaggregationintermediates. Inthisvein,theHanlabdiscoveredthatRNAinducesliquid-liquidphaseseparationofTauinvitrointoprotein droplets held together by weak electrostatic forces. At the in vivo cellular level, the Kosik lab discovered Tau- tRNAcomplexes,therebyaddingTautothegrowinglistofRNA-bindingproteinsinvolvedinneurodegeneration, andcapableofestablishingliquid-liquidphaseseparationinthecytoplasm.TheTau-tRNAcomplexesmaybea physiologic or pathological entity?we will obtain clues by determining their loci in neuronal cells. Finally, we intend to learn whether the conformation of Tau, as modulated by disease mutations or co-factors, influences thestabilityandinvivolocalityoftheTau-tRNAcomplexes.Ourgoalistodiscoveradetailedroutefromsoluble Tautofibrils,fromthenanometertothecellularlevel,anddiscoverthepathologicalentitiesofTauaggregation.